Presence Of GSSG Research Articles

Modeling the stimulation by glutathione of the steady state kinetics of an adenosine triphosphate binding cassette transporter.

We report the steady state ATPase activities of the ATP Binding Cassette (ABC) exporter NaAtm1 in the absence and presence of a transported substrate, oxidized glutathione (GSSG), in detergent, nanodiscs, and proteoliposomes. The steady state kinetic data were fit to the “nonessential activator model” where the basal ATPase rate of the transporter is stimulated by GSSG. The detailed kinetic parameters varied between the different reconstitution conditions, highlighting the importance of the lipid environment for NaAtm1 function. The increased ATPase rates in the presence of GSSG more than compensate for the modest negative cooperativity observed between MgATP and GSSG in lipid environments. These studies highlight the central role of the elusive ternary complex in accelerating the ATPase rate that is at the heart of coupling mechanism between substrate transport and ATP hydrolysis.

Inhibition of glutathione reductase uncovers the activation of NADPH‐inhibited glucose‐6‐phosphate dehydrogenase

Eggleston and Krebs pointed to a paradox in glucose-6-phosphate dehydrogenase (G6PD) regulating process that has not yet been solved, and which originated the term "fine regulation" of G6PD and, therefore, of oxidative phase of pentose phosphate pathway (OPPP). The paradox is that, in basal-like conditions, the activity of G6PD evaluated "in vitro" is very low or nearly null because of the potent inhibiting effect exerted by NADPH, a coenzyme whose concentration in the cell is much higher than that of the substrate NADP+ . However, "in vivo," flow through OPPP occurs in basal conditions. Eggleston and Krebs speculated on the possible existence of a system that would reverse the inhibition by NADPH. Such system would involve oxidized glutathione and exclude the participation of glutathione reductase (GR). The present work confirms the experimental results obtained by Eggleston and Krebs and proves that oxidized glutathione (GSSG) in the absence of NADPH is a direct inhibitor of G6PD. In the presence of GSSG, the G6PD activity recovery system suggested can be observed when GR is previously inhibited by alkylating agents. An unknown element with a molecular weight ranging between 12 and 50kDa has been found to reverse part of G6PD inhibition by NADPH.

Measurement of the tripeptides glutathione and ophthalmic acid by gas chromatography-mass spectrometry

Glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is the most abundant intracellular dicarboxylic tripeptide with multiple physiological roles. GSH and its symmetric disulfide GSSG co-exist in biological samples. The GSH-to-GSSG molar ratio is high within cells and low in extracellular compartments. It is widely used as a biomarker of oxidative stress. Here, we report on a novel two-step derivatization procedure which allows for the specific quantitative measurement of GSH by gas chromatography-mass spectrometry (GC-MS) in the electron-capture negative-ion chemical ionization mode. The method is based on the derivatization of GSH first with pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C) and subsequently with 2 M HCl/CH3OH (30 min, 80 °C). Esterification in 2 M HCl/CD3OD is used for the in situ synthesis of deuterium-labelled GSH for use as internal standard. Quantification is performed by selected-ion monitoring of m/z 557 for GSH and m/z 560 for the internal standard (1 mM). Linear regression analysis between the peak area ratio of m/z 557 to m/z 560 (y) and the GSH concentration (x; range, 0–1 mM) resulted in the regression equations y = 0.02 + 0.62x (r2 = 0.976) in the absence and y = 0.02 + 0.63x (r2 = 0.997) in the presence of GSSG (range, 0–1 mM), underlying the linearity of the method for GSH and suggesting lack of interference by GSSG. γ-Glu-Cys and Cys-Gly also did not interfere. The GC-MS method demonstrated that in human hemolysate, nitrite at toxicological concentrations (range, 0–15 mM) depletes erythrocytic GSH. For the GC-MS analysis of the GSH tripeptide analogue ophthalmic acid, which lacks a Cys moiety, the derivatization order was HCl/CH3OH followed by pentafluoropropionic anhydride in ethyl acetate.

Ultra-Rapid Glutathionylation of Ribonuclease: Is this the Real Incipit of its Oxidative Folding?

Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40–50 times higher reactivity toward GSSG. This phenomenon is partially due to a low pKa value of most of these cysteines (average pKa = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex (KD = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5’-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease.

Altered Glutathione Homeostasis in NLRP3 Inflammasome Activation Mediated by ATP-P2X7 Receptor Signaling

NLRP3 inflammasome is an intracellular protein complex of which activation is responsible for maturation of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-18. Full activation of NLRP3 inflammasome requires two distinct steps; expression of component proteins as well as pro-IL-1beta induced by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), followed by complex formation activated by damage-associated molecular patterns (DAMPs) such as ATP. Although redox-dependent regulation of the complex formation has been suggested, it remains unclear how cellular redox status is regulated during ATP stimulation in cells. Here we describe that glutathione (GSH), an antioxidant peptide abundantly present in cells, plays an important role in ATP-dependent inflammasome activation. LC-MSMS analyses clearly demonstrated that cellular levels of GSH were rapidly decline upon ATP stimulation in LPS-primed mouse macrophage J774.1 cells. Similar levels of GSH were detected in the culture supernatants suggesting that ATP stimulated GSH efflux. It should be noted that ATP treatment also induced efflux of polysulfurated derivatives of GSH such as GSSH and GSSSH as well. P2X7 receptor antagonist A-804598 treatment remarkably inhibited both IL-1beta production as well as decrease of GSH. GSH or its oxidized form GSSG added extracellularly suppressed ATP-dependent decline of intracellular GSH, and more importantly, IL-1beta production. Analyses with use of ROS-sensitive probe DCDHF-DA revealed that ATP stimulation augmented ROS production in macrophages, and it was cancelled in the presence of GSSG. Thus, macrophages use ATP-P2X7 receptor signaling to modulate GSH homeostasis via GSH efflux, thereby regulating redox balance for the inflammasome activation.

Glutathione modifies the oxidation products of 2′-deoxyguanosine by singlet molecular oxygen

The oxidation of the free nucleoside 2′-deoxyguanosine (dGuo) by singlet molecular oxygen (1O2) has been studied over the three last decades due to the major role of DNA oxidation products in process such as ageing, mutation and carcinogenesis. In the present work we investigated the dGuo oxidation by 1O2 in the presence of the important low molecular antioxidant, glutathione, in its reduced (GSH) and oxidized (GSSG) forms. There were applied different conditions of concentration, pH, time of incubation, and the use of a [18O]-labeled thermolabile endoperoxide naphthalene derivative as a source of [18O]-labeled 1O2. Data was obtained through high performance liquid chromatography (HPLC) and HPLC coupled to micrOTOF Q-II analysis of the main oxidation products: the diastereomers of spiroiminodihydantoin-2′-deoxyribonucleosides (dSp) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). An intriguing result was that 8-oxodGuo levels increased by 100 fold when dGuo was oxidized by 1O2 in the presence of GSH and by 2 fold in the presence of GSSG, while dSp levels dropped to zero for both conditions. All data from dGuo, 8-oxodGuo and dSp quantification together with the analysis of residual GSH/GSSG content in each sample strongly suggest that glutathione modifies the mechanism of dGuo oxidation by 1O2 by disfavoring the pathway of dSp formation.

Adding glutathione to parenteral nutrition prevents alveolar loss in newborn Guinea pig

Bronchopulmonary dysplasia, a main complication of prematurity, is characterized by an alveolar hypoplasia. Oxidative stress is suspected to be a trigger event in this population who has a low level of glutathione, a main endogenous antioxidant, and who receives high oxidative load, particularly ascorbylperoxide from their parenteral nutrition. Hypothesis: the addition of glutathione (GSSG) in parenteral nutrition improves detoxification of ascorbylperoxide by glutathione peroxidase and therefore prevents exaggerated apoptosis and loss of alveoli.Methods: Ascorbylperoxide is assessed as substrate for glutathione peroxidase in Michaelis-Menten kinetics. Three-days old guinea pig pups were divided in 6 groups to receive, through a catheter in jugular vein, the following solutions: 1) Sham (no infusion); 2) PN(-L): parenteral nutrition protected against light (low ascorbylperoxide); 3) PN(+L): PN without photo-protection (high ascorbylperoxide); 4) 180μM ascorbylperoxide; 5) PN(+L)+10μM GSSG; 6) ascorbylperoxyde+10μM GSSG. After 4 days, lungs were sampled and prepared for histology and biochemical determinations. Data were analysed by ANOVA, p<0.05Results: The Km of ascorbylperoxide for glutathione peroxidase was 126±6μM and Vmax was 38.4±2.5nmol/min/ U. The presence of GSSG in intravenous solution has prevented the high GSSG, oxidized redox potential of glutathione, activation of caspase-3 (apoptosis marker) and loss of alveoli induced by PN(+L) or ascorbylperoxide.Conclusion: A correction of the low glutathione levels observed in newborn animal on parenteral nutrition, protects lungs from toxic effect of ascorbylperoxide. Premature infants having a low level of glutathione, this finding is of high importance because it provides hope in a possible prevention of bronchopulmonary dysplasia.

Influence of glutathione on the bioactivity of subcutaneously or orally administered insulin to rats.

The effect of reduced (GSH) and oxidized (GSSG) glutathione on the bioactivity of insulin was studied. A polyelectrolyte complex (PEC) of insulin with low molecular weight chitosan (13 kDa) was prepared and characterized. The PEC was then solubilized, in the presence and absence of GSH and GSSG, in a reverse micelle consisting of oleic acid and two surfactants (PEG-8 caprylic/capric glycerides and polyglycerol-6-dioleate). The in vitro and in vivo performances of the reverse micelle formulations (RMFs) were evaluated in rats. At pH 6.5 the association efficiency of the PEC was 76.2%. In vitro insulin release from the RMs was negligible at pH 1.2 and was markedly increased at pH 6.8. The hypoglycemic activity of insulin in the PEC was reduced when administered via the subcutaneous route, regardless of the GSH content. On the other hand, the presence of GSSG significantly enhanced hypoglycemia. When the RMF was administered via the oral route, the presence of GSH had no effect on the hypoglycemic activity of insulin compared with the GSH free system. However, the presence of GSSG in the oral preparation increased the hypoglycemic activity of insulin; probably by inhibiting insulin degradation, thereby prolonging its effect. Thus, incorporation of GSSG in the RMF reduces blood glucose levels in rats and protects insulin from degradation.

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca2+-induced Ca2+ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca2+ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca2+ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca2+ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca2+]SR, increased SR Ca2+ fractional release, and increased spark- and non-spark-mediated SR Ca2+ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca2+ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca2+ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca2+ handling during periods of oxidative stress.

A sensitive and selective chemosensor for GSSG detection based on the recovered fluorescence of NDPA-Fe3O4@SiO2-Cu(II) nanomaterial

A sensitive and selective sensor for oxidized glutathione (GSSG) detection based on the recovered fluorescence of naphthalimide-DPA (NDPA)-Fe3O4@SiO2-Cu(II) system is reported. NDPA-Fe3O4@SiO2 was characterized by X-ray power diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectra (FT-IR) and fluorophotometry. The fluorescence of NDPA-Fe3O4@SiO2 could be quenched by Cu2+ due to the coordination of Cu2+ with the tridentate receptor DPA. This coordination process reduced the electron-donating ability of the nitrogen atom in the DPA moiety, thus suppressing the internal charge transfer (ICT) process in NDPA-Fe3O4@SiO2. In the presence of GSSG, the fluorescence of NDPA-Fe3O4@SiO2-Cu(II) was recovered because of strong coordination of Cu2+ with GSSG, which promoted the decomplexation between NDPA-Fe3O4@SiO2 and Cu2+, and enhanced the ICT process. The NDPA-Fe3O4@SiO2-Cu(II) nanomaterial exhibited high sensitivity towards GSSG, and a good linear relationship was obtained from 5nM to 60μM. The limit of detection, based on a signal-to-noise ratio of 3, was 50pM. In addition, the presence of magnetic Fe3O4 nanoparticles (NPs) in NDPA-Fe3O4@SiO2 NPs would also facilitate the magnetic separation of NDPA-Fe3O4@SiO2 from the solution. Through the use of added internal standards, we successfully determined the concentration of GSSG in HEK 293 cell lysate to be 1.15μM by the prepared chemsensor NDPA-Fe3O4@SiO2-Cu(II). The proposed method is anticipated to fabricate other sensitive fluorescence sensors based on organic–inorganic hybrid magnetic nanoparticles.

Oxidative Modification of Rat Sulfotransferase 1A1 Activity in Hepatic Tissue Slices Correlates with Effects on the Purified Enzyme

Mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of xenobiotics as well as numerous endogenous molecules. The major aryl (phenol) SULT in rat liver, rSULT1A1, has been used extensively as a model enzyme for understanding the catalytic function of SULTs. Previous studies showed that purified rSULT1A1 displays significant catalytic changes in the presence of GSSG and other oxidants. In the present study, the effects of diamide [1,1'-azobis(N,N-dimethylformamide)] and tert-butyl hydroperoxide (TBHP) on the activity of rSULT1A1 in rat hepatic slices were compared with the effects of these oxidants on a homogeneous preparation of the enzyme. Precision-cut hepatic slices were incubated with 10 μM 7-hydroxycoumarin (7-HC) in the presence of varied concentrations of either diamide or TBHP. Analysis of the 7-hydroxycoumarin sulfate released into the incubation medium indicated that both oxidants significantly increased the sulfation of 7-HC, and this occurred at optimal concentrations of 5 and 10 μM, respectively. Cellular GSH and GSSG levels in the hepatic slices were not significantly altered from control values at these concentrations of diamide and TBHP. Exposure of homogeneous rSULT1A1 to diamide or TBHP also increased the rate of sulfation of 7-HC, although the optimal concentrations of diamide and TBHP were lower (50- and 100-fold, respectively) than those required for effects with the hepatic slices. These results indicate that both diamide and TBHP may modify the rSULT1A1 in intact cells in a manner similar to that observed with the homogeneous purified enzyme.

Redox dependence of transport activity of tonoplast proton pumps: Effects of nitric oxide exposure during ontogenesis and under hypoosmotic and hyperosmotic stress

Variations of the redox status is shown to inhibit the transport activity of tonoplast proton pumps at different stages of ontogenesis and under the conditions of hyperosmotic stress. However, the activity of H+-ATPase increased by 60% under hypoosmotic stress in the presence of GSH. The influence of nitric oxide on the transport activity of tonoplast proton pumps also depended on the redox status. In the case of change of the redox status, stimulating effect of nitric oxide turned inhibitory, except for simultaneous application of hypoosmotic stress and nitric oxide. In this case, stimulation of both proton pumps was observed and the activity of H+-ATPase increased in the presence of GSH, though the activity of H+-PPase increased in the presence of GSSG. This may explain the necessity of the presence in the vacuolar membrane of two proton pumps having similar functions.

Synthesis, characterization and fluorescent properties of cerium(III) glutathione complex

The cerium (III) glutathione complex was synthesized by the redox reaction of cerium (IV) with glutathione reduced (GSH) in aqueous solution. The Job-plots indicate an ML (L = GSSG) stoichiometry of the complex. The fluorescent properties of the compound were investigated. The as-prepared complex showed the characteristic maximum emission spectra of Ce(III) at 350 nm (λ(ex) = 255 nm). The fluorescence results show that the Ce(IV) ions are first reduced to Ce(III), and then form Ce(III) complex after reacting with GSH. The complex was characterized by element analysis and FT-IR spectra; the stability of the complex was analyzed by cyclic voltammeters and DSC-TG as well. Finally, Ce(IV) was successfully employed to determine the concentrations of GSH in the presence of GSSG, in which the fluorescence intensities are proportional to the concentrations of GSH in the range of 1-100 nM with the detection limit of 0.05 nM of GSH, without interference from the presence of GSSG.

Ozone Oxidizes Glutathione to a Sulfonic Acid

Biosurfaces are universally covered with fluid microfilms containing reduced glutathione (GSH) and other antioxidants whose putative roles include the detoxification of ambient ozone (O(3)). It is generally believed that O(3) accepts an electron from the thiolate GS(2-) function [pK(a)(GS(-)) = 8.8] of GSH to produce thiyl GS(*-) radicals en route to the disulfide GSSG. Here, we report novel electrospray mass spectrometry experiments showing that sulfonates (GSO(3)(-)/GSO(3)(2-)), not GSSG, are the exclusive final products on the surface of aqueous GSH microdroplets exposed to dilute O(3)(g) for approximately 1 ms. The higher reactivity of the thiolate GS(2-) toward O(3)(g) over the thiol GS(-) is kinetically resolved in this time frame due to slow GS(-) acid dissociation. However, our experiments also show that O(3) will be largely scavenged by the more reactive ascorbate coantioxidant in typical interfacial biofilms. The presence of GSSG and the absence of GSO(3)(-)/GSO(3)(2-) in extracellular lining fluids are therefore evidence of GSH oxidation by species other than O(3).

Electrochemical Study of Methyl 2-[p-Nitrophenyl(hydroxy)methyl]acrylate: An Anticancer Drug and Its Reactivity Toward GSH and Oxygen

Electrochemical experiments with methyl 2-[-nitrophenyl(hydroxy)methyl]acrylate were performed in protic (, , pH 6.9; , or , pH 9.4 and , , pH 9.6) and aprotic media. The primary reduction behavior in aprotic medium was typical of nitroaromatics along with an additional wave related to the reduction of the acrylate function. Kinetic analysis carried out in aprotic and aqueous basic media pointed out to the high stability of the electrogenerated nitro radical anion, especially in . Reduced (GSH) and oxidized (GSSG) gluthatione in phosphate buffer influenced the reduction behavior of , due mainly to protonation effects. Direct reduction of , in the presence of GSH, led to a transient nitroso-GS adduct. In the presence of GSSG, hydrogen-bonding-associated GSSG-hydroxylamine was the main product. Electrochemical studies of , in the presence of oxygen, showed no chemical reactivity between and . These electrochemical results help in the understanding of the anticancer activity of that can be considered a bioreductive agent with a glutathione depleting function.

Identification of a Novel Glutathione Conjugate of Flutamide in Incubations with Human Liver Microsomes

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.

Functional properties and oxidative modulation of A‐type K+ currents in hippocampal granule cells of control and chronically epileptic rats

A-type K+ channels are crucial determinants of neuronal firing. For example, reducing the amplitude of A-type currents (I(A)) increases seizure susceptibility. We have therefore examined the functional and molecular properties of I(A) in dentate granule neurons following pilocarpine-induced status epilepticus (SE). We found that the levels of various A-type channel subunit mRNAs are unaltered following SE. Furthermore, current density and biophysical properties of I(A) recorded in outside-out and cell-attached patches from dentate granule cells are not modified by SE. However, I(A) in both control and epileptic rats was powerfully regulated by the cellular redox state. I(A) was recorded in outside-out patches with the recording pipette containing either reduced (GSH) or oxidized (GSSG) glutathione. In both control and epileptic rats, the presence of GSSG caused a similar, marked acceleration of recovery from inactivation. Additionally, GSSG produced a small but significant reduction of I(A) amplitudes only in control rats. The inactivation time course of I(A) during depolarizing voltage steps was not modified by GSH or GSSG. Cell-attached recordings, in which the intracellular milieu is conserved, revealed a slow time course of recovery more comparable to that with GSH. In summary, epileptic activity does not produce chronic changes in the molecular and functional properties of the somatic I(A) of dentate granule cells. However, I(A) is powerfully modulated by oxidation in both control and epileptic rats. This finding suggests that the availability of I(A) may be strongly regulated by changes in the GSH/GSSG ratio occurring during prolonged seizure activity or hypoxia.

The N-terminal Domain of PILB from Neisseria meningitidis Is a Disulfide Reductase That Can Recycle Methionine Sulfoxide Reductases

The PilB protein of the Neisseria genus comprises three domains. Two forms have been recently reported to be produced in vivo. One form, containing the three domains, is secreted from the bacterial cytoplasm to the outer membrane, whereas the second form, which is cytoplasmic, only contains the central and the C-terminal domains. The secreted form was shown to be involved in survival under oxidative conditions. Although previous studies indicated that the central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively, no function was described so far for the N-terminal domain. In the present study, the N-terminal domain of the PilB of Neisseria meningitidis was produced as a folded entity, and its biochemical and enzymatic properties have been determined. The data show that the N-terminal domain possesses a disulfide redox-active site with a redox potential in the range of that of thioredoxin. Moreover, the N-terminal domain, either as an isolated form or included in PilB, recycles the oxidized forms of the methionine sulfoxide reductases like thioredoxin. These results, which show that the N-terminal domain exhibits a disulfide reductase activity and probably has a thioredoxin-fold, are discussed in relation to its possible functional role in Neisseria.

Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin

An open reading frame of gelonin (Gel), one of ribosome inactivating proteins, was inserted into the vector pBSL-C which contains the coding region of chitin binding domain (CBD)-intein, resulting in the fusion expression of CBD-intein–Gel in Escherichia coli BL21 (DE3) by the induction of IPTG. The fusion product formed an aggregate of the misfolded protein, commonly referred to as inclusion bodies (IBs). The IBs were denatured and then refolded by step-wise dialysis. About 69% fusion protein was in vitro refolded to native state in the presence of GSSG and GSH as monitored by size-exclusion HPLC. The refolded CBD-intein–Gel was loaded onto chitin beads column equilibrated with 10 mM Tris buffer, 500 mM NaCl, pH 8.5, and about 2.4 mg Gel/L culture with 96% homogeneity was directly eluted from the captured column by incubation at 25 °C under pH 6.5 for 48 h based on intein C-terminal self-cleavage. Western blot, ELISA, and in vitro inhibition of protein synthesis demonstrated that the bioactivity of recombinant Gel was comparable to that of native Gel purified from seeds. This implied that the purified Gel by this method is biologically active and suitable for further studies.

A novel function of tissue-type transglutaminase: protein disulphide isomerase.

We have found that tissue-type transglutaminase (tTG), also called TGc, TGase2 and Galpha(h), has the activity of protein disulphide isomerase (PDI). We have shown that tTG converts completely reduced/denatured inactive RNase A molecule to the native active enzyme. The PDI activity of tTG was strongly inhibited by bacitracin, which is a frequently used inhibitor of conventional PDI activity. It was substantially inhibited by the simultaneous presence of other potential substrate proteins such as completely reduced BSA, but not by native BSA. This activity was especially high in the presence of GSSG, but not GSH. The addition of GSH to the reaction mixture in the presence of GSSG at a fixed concentration up to at least 200-fold excess did not very substantially inhibit the PDI activity. It is possible that tTG can exert PDI activity in a fairly reducing environment like cytosol, where most of tTG is found. It is quite obvious from the following observations that PDI activity of tTG is catalysed by a domain different from that used for the transglutaminase reaction. Although the alkylation of Cys residues in tTG completely abolished the transglutaminase activity, as was expected, it did not affect the PDI activity at all. This PDI activity did not require the presence of Ca(2+). It was not inhibited by nucleotides including GTP at all, unlike the other activity of tTG.